CN104769110A - Nucleic acid analysis kit and nucleic acid analysis method - Google Patents

Nucleic acid analysis kit and nucleic acid analysis method Download PDF

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CN104769110A
CN104769110A CN201480002850.3A CN201480002850A CN104769110A CN 104769110 A CN104769110 A CN 104769110A CN 201480002850 A CN201480002850 A CN 201480002850A CN 104769110 A CN104769110 A CN 104769110A
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nucleic acid
probe
step
acid analysis
analysis
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CN201480002850.3A
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堀邦夫
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奥林巴斯株式会社
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Priority to PCT/JP2014/062917 priority patent/WO2014188941A1/en
Publication of CN104769110A publication Critical patent/CN104769110A/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813Hybridisation assays
    • C12Q1/6834Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
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    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813Hybridisation assays
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813Hybridisation assays
    • C12Q1/6834Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844Nucleic acid amplification reactions

Abstract

The present invention accurately identifies the respective origins of multiple samples that are submitted for analysis using a small number of nucleic acid species. Provided is a nucleic acid analysis kit (100), which is equipped with multiple supports (2) for holding a nucleic acid-containing identification probe (1) and supporting a sample. In addition to the identification probe (1) comprising at least one kind of nucleic acid with a known base sequence, the variety and/or the amount of the nucleic acid differs for each support (2) and the identification probe is held in the support (2) so as to be miscible with a sample.

Description

核酸分析试剂盒及核酸分析方法 The nucleic acid analysis kit and nucleic acid analysis method

技术领域 FIELD

[0001] 本发明涉及一种核酸分析试剂盒及核酸分析方法。 [0001] The present invention relates to a kit for nucleic acid analysis and nucleic acid analysis method.

背景技术 Background technique

[0002] 以往,在核酸分析中,从提取待检体到供于分析该待检体中所含有的核酸期间,在进行从待检体提取核酸等前处理中进行多次容器的交换。 [0002] Conventionally, in nucleic acid analysis, the sample to be extracted from the analysis period for the nucleic acid to be contained in the specimen, a plurality of times during the exchange vessel and nucleic acid extraction from a pre-treatment to be subject. 通常,在以手工操作进行前处理的情况下,通过每进行一次容器交换时对交换前后的容器进行对应,从而防止样品取错。 Typically, in the case of manual operation prior to processing, once the container before and after the exchange of the container through the corresponding exchange for each, so as to prevent the wrong sample taken. 在待检体数目很多的情况下,进行容器的对应操作是很繁琐的,且发生样品取错的概率变高。 When the number of the subject to be a lot of cases, the corresponding operation is very cumbersome container, and the probability of occurrence of error samples taken increases.

[0003] 作为防止该样品取错的方案,已知有将识别子探针混合于待检体中,将识别子探针与待检体一起进行扩增的方法(例如,参照专利文献I。)。 [0003] As the sample is taken to prevent the wrong program, there is known to be mixed with the probes to identify the sub-sample, the probe will identify the sub-body to be detected with the amplification method (e.g., refer to Patent Document I. ). 该识别子探针在通过与待检体相同的引物而被扩增的区域具有可对于附于各待检体中的个别编码进行解码的碱基序列。 The promoter probe having a base sequence recognition can be decoded for the subject to be attached to each of the individual coding regions are to be amplified by the same primer subject. 因此,在扩增待检体后,通过对扩增产物中所含有的识别子探针的个别编码进行解码,从而可特定扩增产物源自于哪个待检体。 Thus, to be detected after amplification thereof, by decoding the identification of the individual sub-coding amplification product contained in the probe, which can be specific amplification product derived from a subject to which.

[0004] 另一方面,作为对于生物组织的切片中所含有的特定的分子的量或活性的空间分布进行解析的方法,已知有将识别靶分子的探针导入至切片上的多个位置,并将识别各探针的切片上的位置的标记物(Tag)附于该探针的方法(例如,参照专利文献2。)。 [0004] On the other hand, as a method for the amount of a particular molecule of a biological tissue slice contained in the active or resolved spatial distribution, there is known a plurality of positions on the identified target molecules introduced into the probe slice and the position of the marker on the probe identifying each slice (tag) is attached to the probe method (e.g., refer to Patent Document 2). 从切片中回收与分子反应的探针,并分析附于探针的标记物,从而可知切片的各位置中的靶分子的量或活性。 Recovered from the slices of the reaction with the probe molecule, and analyze markers attached to the probe, so that the amount or activity found in each position in the slice of the target molecule.

[0005] 现有技术文献 [0005] The prior art documents

[0006] 专利文献 [0006] Patent Document

[0007] 专利文献1:日本特开2007-74967号公报 [0007] Patent Document 1: Japanese Laid-Open Patent Publication No. 2007-74967

[0008] 专利文献2:美国专利申请公开第2011/0245111号说明书 [0008] Patent Document 2: U.S. Patent Application Publication No. 2011/0245111 specification

发明内容 SUMMARY

[0009] 发明要解决的问题 [0009] Problems to be solved

[0010] 然而,专利文献I及2中记载的方法中,对于每个待检体需要准备具有不同碱基序列的识别子探针或标记物。 [0010] However, in Patent Documents I and 2 in the method described, for each specimen to be prepared has a need to identify promoter probe or marker different base sequences. 即,在分析对象的待检体数目很多的情况下,有对于不得不准备很多种的识别子探针或标记物的不便。 That is, when the number of objects to be examined analyzer many cases, there are very inconvenient to have to prepare probes or more of the sub-identification markers.

[0011] 本发明是鉴于上述问题而完成的,其目的在于提供一种利用很少种类的核酸就能很准确地鉴定供于分析的多个样品的各自的来源的核酸分析试剂盒及核酸分析方法。 [0011] The present invention has been accomplished in view of the above problems, and its object is to provide a kind of a nucleic acid with few assay kit for nucleic acid analysis and analyzing a plurality of samples of each of the source can be very accurate identification of method.

_2] 用于解决问题的方案 _2] Solution to Problem

[0013] 为了实现上述发明目的,本发明提供以下的方法。 [0013] In order to achieve the above object, the present invention provides the following method.

[0014] 本发明的第I方式是一种核酸分析试剂盒,其具备多个支撑体,所述多个支撑体保持含有核酸的识别探针且支撑待检体,前述识别探针含有具有已知的碱基序列的至少I种核酸且每个前述支撑体中前述核酸的种类和量中的至少一者不同并以能够混合于前述待检体的方式保持在前述支撑体。 [0014] I first embodiment of the present invention is a kit for nucleic acid analysis, comprising a plurality of supports, the plurality of support holder comprising a nucleic acid probe to identify and support member to be detected, has been the identification probe comprising at least one of at least two different types of I type and amount of nucleic acid in the nucleic acid and each of the support body of the known nucleotide sequence and can be mixed in the manner to be held in the specimen support.

[0015] 根据本发明的第I方式,在使待检体分别支撑于多个支撑体待检体时,保持在支撑体的识别探针中所含有的检测用的核酸混合于待检体中。 [0015] According to a first embodiment I of the present invention, when the body to be examined are supported by the support member to be a plurality of the subject, holding in the recognition nucleic acid detection probe of the support contained in a specimen to be mixed in . 此时在混合于各待检体中的识别用的核酸的种类和量中至少一者,在每个待检体中相互不同。 At this time, the type and amount of nucleic acid to be mixed in each of the sample recognition of at least one, to be different from each other in each of the subject. 因此,在对由支撑于支撑体的各待检体所制备的样品进行核酸分析时,通过对每个样品中所含有的识别用的核酸也进行解析,且对该核酸的种类和/或量也进行鉴定,从而可以基于该检测用的核酸的种类和/或量很准确地鉴定各样品源自于哪个待检体。 Thus, when each of the samples prepared by the specimen to be supported by the support body for nucleic acid analysis, and analyzed by the identification of a nucleic acid contained in each sample, and the nucleic acid type and / or amount of also identified, can be based on the kind of the nucleic acid detection and / or amount of each sample was accurately identified to be from the subject in which.

[0016] 如此,通过使混合于各待检体中的识别用的核酸的种类和量中的至少一者不同,从而可以进行多个待检体相互间的识别,因此识别探针只要含有很少种类的核酸即可,从而能够利用很少种类的核酸来准确地鉴定多个样品的各自的来源。 [0016] Thus, the kind and amount of nucleic acid by mixing the respective body to be examined with the identification of at least one different, so that each can be identified among the plurality of bodies to be detected, so long as it contains the probe is identified nucleic acid type can be small, it is possible to accurately identify the source of each of the plurality of samples with few kinds of nucleic acids.

[0017] 上述第I方式中,可以具备对应表,所述对应表将前述多个支撑体与分别被该多个支撑体保持的前述识别探针的组成即前述核酸的种类和量的组合相对应。 [0017] In the first embodiment I, may be provided with the correspondence table, the correspondence table identifying the composition of the probe and the plurality of supports are held by the plurality of support that is a combination of the kind and amount of the nucleic acid with correspond.

[0018] 以此方式,可以很容易地把握在哪个支撑体上保持有哪个组成的识别探针。 [0018] In this manner, you can easily grasp the support on which the probe identified which holds thereof.

[0019] 上述第I方式中,前述支撑体为收容前述待检体的容器,前述识别探针也可以被封入至前述容器内。 [0019] In the first embodiment I, the support is a container accommodating the specimen to be, the identification probe can also be sealed in the inner container.

[0020] 以此方式,仅向容器内投入待检体就可以很容易地将识别探针混合于待检体中。 [0020] In this manner, only to be put into the specimen container can be easily identified to be mixed in the sample probe body.

[0021] 上述第I方式中具备基板,所述基板能够沿着规定的分割线分割成多个芯片且粘贴有作为前述待检体的生物组织的切片,前述识别探针可分别被前述多个芯片保持。 [0021] In the first embodiment I comprises a substrate along a predetermined dividing line can be divided into a plurality of chips and the biological tissue is attached sections as the body to be examined, the identification of the plurality of probes may be respectively chip holding.

[0022] 以此方式,在对作为待检体的生物组织的切片的一部分进行分析的情况下,通过将切片粘贴于基板后并沿着分割线将基板和切片同时分割,从而可制备混合了一种识别探针的切片的片段。 [0022] In this manner, in the case where the subject as part of a biological tissue slice to be analyzed by the sections and attached to the substrate and the sections divided along the dividing line while the rear substrate, thereby preparing mixed a method of identifying fragments of the probe slice.

[0023] 上述第I方式中,前述核酸可以为DNA (脱氧核糖核酸)。 [0023] In the first embodiment I, the nucleic acid may be a DNA (deoxyribonucleic acid).

[0024] 以此方式,可制成为适合于待检体中所含有的DNA的分析的构成。 [0024] In this manner, the configuration may be made to be suitable for the DNA contained in the sample analysis.

[0025] 上述第I方式中,前述核酸可以为RNA(核糖核酸)。 [0025] In the first embodiment I, the nucleic acid may be RNA (ribonucleic acid).

[0026] 作为待检体的分析对象的核酸为RNA时,识别探针中所含有的核酸优选为RNA。 [0026] When the object to be analyzed as a nucleic acid sample is RNA, the nucleic acid probe to identify is preferably contained in the RNA. 在识别探针含有RNA时,为了不被RNA核糖核酸酶分解,也可使用被修饰的RNA例如寡核苷酸的2'位置取代为甲基的RNA等。 Upon identifying RNA probe contains, in order not to be decomposed ribonuclease RNA, may be used, for example, modified RNA oligonucleotide 2 'position is substituted with a methyl RNA and the like.

[0027] 上述第I方式中,前述核酸可以为核酸类似物质。 [0027] In the first embodiment I, the nucleic acid may be a nucleic acid analog. 作为核酸类似物质,可以举出:如DNA或RNA这样的天然型核苷酸(天然存在的核苷酸)的侧链等被氨基等官能团修饰的物质、或者被蛋白质或低分子化合物等标识的物质。 As the nucleic acid-like substance may include: identifying a modified material, such as an amino functional group such as DNA or RNA such natural type nucleotide (naturally occurring nucleotides) side chains and the like, or low molecular compound is a protein or the like substance. 更具体地,例如可举出:桥联的核酸(Bridged nucleic acid:BNA)、2,-O, 4,-C-乙稀桥联核酸(2,-O, 4,-C-Ethylene-bridgedNucleic Acids:ENA)、天然型核苷酸的4'位氧原子被硫原子取代的核苷酸、天然型核糖核苷酸的2'位轻基被甲氧基取代的核苷酸、己糖醇核酸(Hexitol Nucleic Acid:HNA)、肽核酸(PNA)等。 More specifically, for example, include: bridged nucleic acid (Bridged nucleic acid: BNA), 2, -O, 4, -C- ethylene-bridged nucleic acids (2, -O, 4, -C-Ethylene-bridgedNucleic Acids: ENA), naturally-occurring nucleotide 4 'position is substituted with an oxygen atom a sulfur atom nucleotides, naturally-occurring ribonucleotide 2' nucleotide position light methoxy group substituted hexitol nucleic acid (Hexitol nucleic Acid: HNA), peptide nucleic acid (PNA) and the like.

[0028] 上述第I方式中,前述DNA的碱基长度可为20个碱基以上且500个碱基以下。 [0028] In the first embodiment I, the DNA base length may be 20 bases or more and 500 or less bases.

[0029] 以此方式,可使识别探针的制备及在核酸分析或前处理过程中的DNA操作更加容易O [0029] In this manner, DNA can be prepared and the operation recognition probes in nucleic acid analysis or during pre-treatment easier O

[0030] 上述第I方式中,保持在各前述支撑体的前述DNA的分子数可为100分子以上且100000分子以下。 [0030] In the first embodiment I, the number of DNA molecules maintained in each of the support body 100 may be a molecule or more and 100,000 or less molecule.

[0031] 以此方式,识别探针不会对于待检体中所含有的核酸的分析带来影响,从而能够确保识别探针的DNA的定量精度。 [0031] In this manner, the probe does not affect the identification of nucleic acids for the analysis to be contained in the specimen, thereby ensuring the accuracy of quantification of the DNA recognition probes.

[0032] 本发明的第2实施方式为核酸分析方法,其包括:探针添加工序:将包含具有已知的碱基序列的至少I种核酸的识别探针分别添加至多个待检体中;和核酸分析工序:对于各前述待检体中所含有的核酸进行分析。 [0032] The second embodiment of the present invention is a nucleic acid analysis method, comprising: step of adding a probe: nucleic acid comprising at least I recognition probe having a known base sequence to be detected are added to the plurality of bodies; and a step of nucleic acid analysis: analyzed for each of the nucleic acid to be contained in the specimen. 在前述探针添加工序中,将前述核酸的种类和量中的至少一者相互不同的前述识别探针添加至前述多个待检体中,在前述核酸分析工序中,对于添加至各前述待检体中的前述识别探针中所含有的前述核酸也进行解析。 In the step of adding the probe, the type and amount of the nucleic acids at least one of each different probe is added to the identification of the plurality of bodies to be detected, wherein in the nucleic acid analysis step, for each of the foregoing be added to the nucleic acid in the specimen contained in the identification probe can also be resolved.

[0033] 根据本发明的第2实施方式,探针添加工序中将识别探针添加至待检体之后,核酸分析工序中,在对由该待检体制备的样品进行核酸分析时,通过对于各样品中所含有的检测用的核酸也进行解析,且对于该核酸的种类和/或量也进行鉴定,从而能够很准确地基于该检测用的核酸的种类和/或量来鉴定各样品源自于哪个待检体。 When, after [0033] According to the second embodiment of the present invention, in the step of adding a probe to identify the probe to be added to the sample, a nucleic acid analysis step, the sample prepared by the subject system for the nucleic acid to be analyzed, by for nucleic acid detection in each sample contained also resolved, and for the type of the nucleic acid and / or the amount identified, it is possible to very accurately based on the type and / or amount of nucleic acid of the detection to identify each sample source which to be subject to the self.

[0034] 如此,通过使添加至各待检体的识别用的核酸的种类和量中的至少一者不同,从而可使多个待检体相互地识别,因此,识别探针只要含有很少种类的核酸即可,就能利用很少种类的核酸很准确地鉴定多个样品的各自的来源。 [0034] Thus, by adding at least one of a different kind and amount of nucleic acid to identify each specimen to be used in, thereby allowing the plurality of sample to be identified with each other, and therefore, as long as the probe contains little recognition nucleic acid type can be, can be very accurately identify the source of each of the plurality of samples with few kinds of nucleic acids.

[0035] 上述第2实施方式中,前述探针添加工序中,可以将前述待检体投入至收容前述识别探针的容器内。 [0035] The above-described second embodiment, the step of adding the probe, the sample can be put into the containers housing the identification of the probe.

[0036] 以此方式,仅将待检体投入至容器就可很容易地将识别探针添加至待检体。 [0036] In this manner, only to be put into the specimen container can easily be added to the probe to identify the subject.

[0037] 上述第2实施方式中,前述探针添加工序中进而包括切断工序:在散在并粘贴有如述识别探针的基板上粘贴生物组织的切片,且将粘贴有如述切片的基板与如述切片一起切断成含有一个识别探针的多个芯片,在前述核酸分析工序中,可对于附着于在前述切断工序中得到的前述芯片上的前述切片的一部分进行分析。 [0037] The above-described second embodiment, the probe further comprises adding step cutting step: pasting the bulk of biological tissue like paste on the substrate and said probe to identify sections of the substrate and the like attached to said slice as described slice cut into a plurality of chips with identification comprising a probe, a nucleic acid in the analysis step, analysis can be performed for a portion of the slices on the chip is attached to the cutting obtained in the previous step.

[0038] 以此方式,可以很容易地对存在于切片的各位置上的核酸进行分析。 [0038] In this manner, the nucleic acid may be present in each position in the slice is analyzed easily.

[0039] 上述第2实施方式中,前述探针添加工序中进而包括切断工序:在生物组织的切片上散在并粘贴前述识别探针,并将粘贴有前述识别探针的前述切片切断成含有一个识别探针的多个片段,前述核酸分析工序中,也可对在前述切断工序中切断的前述切片的片段进行分析。 [0039] The above-described second embodiment, the probe further comprises adding step cutting step: biological tissue in the slice and paste dispersed in the identification probes, and identifying the slice is attached the probe to contain a cut a plurality of segment identification probes, the nucleic acid analysis step, the fragments can be analyzed in the foregoing sections is cut in the cutting step.

[0040] 以此方式,可以很容易地对存在于切片的各位置的核酸进行分析。 [0040] In this manner, the nucleic acid may be present in each position of the slice is analyzed easily.

[0041] 上述第2实施方式中,可以在前述切断工序之前进而包括杂交工序:使用具有与靶核酸互补的已知的碱基序列的核酸探针对前述切片进行原位杂交(in situhybridizat1n)。 [0041] The above-described second embodiment, the hybridization may further comprise the step prior to the cutting step: using a nucleic acid probe having a known base sequence complementary to the target nucleic acid in situ hybridization of the slice (in situhybridizat1n).

[0042] 以此方式,在核酸分析工序中可以基于核酸探针检测出靶核酸,并可以提高靶核酸的检测灵敏度。 [0042] In this manner, the nucleic acid analysis step may be based on nucleic acid probes detected a target nucleic acid, and improves the detection sensitivity of the target nucleic acid.

[0043] 上述第2实施方式中,前述核酸分析工序中,可使用定量核酸扩增法或能够进行核酸定量的碱基序列读取装置对前述待检体及前述识别探针中所含有的核酸进行分析。 [0043] The above-described second embodiment, the nucleic acid analysis step, a quantitative nucleic acid amplification method can be used, or the nucleic acid can be quantified on the nucleotide sequence of the reading means and the identification of the subject to be contained in the probe for analysis.

[0044] 以此方式,能够更准确地定量源自各待检体的样品中所含有的核酸。 [0044] In this manner, it is possible to more accurately quantify a nucleic acid sample from each specimen to be contained.

[0045] 上述第2实施方式中,前述识别探针中所含有的前述核酸也可以为DNA。 [0045] The above-described second embodiment, the nucleic acid probe contained in the identification may be a DNA. 前述DNA的碱基长度优选为20个碱基以上且500个碱基以下,各前述识别探针中所含有的前述DNA的分子数优选为100分子以上且100000分子以下。 Preferably the DNA base length of 20 bases or more and 500 bases or less, the identification of each probe contained in the number of molecules of the DNA molecule is preferably 100 or more and 100,000 or less molecule.

[0046] 上述第2实施方式中,前述核酸分析工序中,使用定量核酸扩增法对添加至各前述待检体中的前述DNA进行分析,前述定量核酸扩增法中的前述DNA的扩增率可大致相同。 [0046] The above-described second embodiment, the nucleic acid analysis step, a quantitative nucleic acid amplification method of the DNA was added to each of the sample to be analyzed, the quantitative amplification of nucleic acid amplification method in the DNA rates may be substantially the same. 更优选的是,前述定量核酸扩增法为PCR(聚合酶链式反应)法,各前述DNA的扩增率的差为1.9倍以下,更优选为1.1倍以下。 More preferably, the nucleic acid amplification method of the quantitative PCR (polymerase chain reaction) method, the difference in amplification rates of the respective DNA is 1.9 times or less, more preferably 1.1 times or less.

[0047] 以此方式,能够更准确地定量源自各待检体的样品中所含有的DNA。 [0047] In this way, the DNA sample can be quantitatively derived from each specimen to be contained more accurately.

[0048] 发明的效果 Effect [0048] invention.

[0049] 本发明具有如下效果:利用很少种类的核酸就能够很准确地鉴定供于分析的多个待检体的各自的来源。 [0049] The present invention has the following effects: with few kinds of nucleic acids can be subjected to very accurate identification of a respective plurality of sources to be the subject of analysis.

附图说明 BRIEF DESCRIPTION

[0050] 图1是本发明的第I实施方式中的核酸分析试剂盒的整体结构图。 [0050] FIG. 1 is an overall configuration diagram of nucleic acid analysis I a first embodiment of the present invention in a kit.

[0051]图2是对于使用图1的核酸分析试剂盒的核酸分析方法进行说明的流程图。 [0051] FIG 2 is a flowchart illustrating a method for the analysis of nucleic acid analysis kit using FIG 1 is a nucleic acid.

[0052] 图3是本发明的第2实施方式中的核酸分析试剂盒的整体结构图。 [0052] FIG. 3 is an overall configuration diagram of nucleic acid analysis of the second embodiment of the present invention in a kit.

[0053] 图4A是对于图3的核酸分析试剂盒所具备的基板的使用方法进行说明的图,表示基板分割前。 [0053] FIG. 4A is a diagram for explaining a method for nucleic acid analysis kit of FIG. 3 provided in the substrate, a front substrate is divided.

[0054] 图4B是对于图3的核酸分析试剂盒所具备的基板的使用方法进行说明的图,表示基板分割后。 [0054] FIG. 4B is a diagram for explaining a method for nucleic acid analysis kit of FIG. 3 provided in the substrate, a rear substrate is divided.

[0055] 图5是对于使用图3的核酸分析试剂盒的核酸分析方法进行说明的流程图。 [0055] FIG. 5 is a flowchart illustrating a method for the analysis of nucleic acid analysis kit using FIG. 3 is a nucleic acid.

[0056] 图6是对于本发明的第3实施方式中的核酸分析方法即使用图3的核酸分析试剂盒的另一个核酸分析方法进行说明的流程图。 [0056] FIG. 6 is a nucleic acid analysis method for the third embodiment of the present invention, i.e., nucleic acid analysis method with another nucleic acid analysis kit of FIG. 3 is a flow chart explaining.

具体实施方式 Detailed ways

[0057](第I实施方式) [0057] (Embodiment of I)

[0058] 以下,参照图1及图2对于本发明的第I实施方式中的核酸分析试剂盒100及使用该试剂盒的核酸分析方法进行说明。 [0058] Hereinafter, with reference to FIGS. 1 and 2 will be described with respect to embodiment I of the present invention in a nucleic acid assay kit using the nucleic acid analysis method and 100 of the kit.

[0059] 关于本实施方式中的核酸分析试剂盒100,如图1所示,具备:封入了识别探针I的多个容器(支撑体)2和对应表3,所述对应表3将各容器2上附有的容器号与封于各容器2的识别探针I的组成相对应。 [0059] For the present embodiment, a nucleic acid assay kit 100, shown in Figure 1, includes: a plurality of sealed container identification Probe I (support member) 2 and a correspondence table 3, the respective correspondence table 3 No. 2 container with the container and sealing the container identification to each of the probes I 2 corresponds to the composition.

[0060] 容器2是例如通常在分子生物学实验中所使用的1.5mL的微量离心管。 [0060] The container 2 is, for example, generally in trace amounts 1.5mL used in molecular biology experiments centrifuge tube. 识别探针I在干燥状态下粘贴于容器2的底部的内表面。 Probe I identified in the dry state attached to the inner surface of the bottom of the container 2.

[0061] 识别探针I包含具有相互不同的已知碱基序列的多种核酸。 [0061] I recognition probe comprises a plurality of mutually different known nucleic acid having the nucleotide sequence. 这些多种核酸以每个容器2相互不同的配混比进行混合,识别探针I的组成即识别探针I中所含有的核酸的种类及其量在每个容器2中不同。 The plurality of nucleic acids each container 2 in mutually different mixing ratio were mixed, i.e., the composition I recognition probe to identify the type and amount of nucleic acid probes contained different I in each container 2.

[0062] 在本实施方式中,对作为核酸使用4种DNAl、DNA2、DNA3和DNA4的情况进行说明。 [0062] In the present embodiment, four kinds DNAl use as a nucleic acid, DNA2, DNA3 and DNA4 situation will be described. DNAl、DNA2、DNA3和DNA4以4个阶段各自不同的配混量进行配混。 DNAl, DNA2, DNA3 and DNA4 to four stages with different respective compounding amounts compounded. 即,在本例中能够制备配混4X4X4X4 = 256种具有相互不同配混比的识别探针1,核酸分析试剂盒100所具有的容器2的最大数为256个。 That is, in the present embodiment can be prepared by compounding 4X4X4X4 = 256 Species identification probes having mutually different mixing ratio of 1, the nucleic acid assay kit 100 has a maximum number of containers 2 to 256.

[0063] 需要说明的是,识别探针I中所含有的核酸的种类可根据待检体的靶核酸的种类进行适宜地选择,也可使用RNA等其他种类的核酸。 [0063] Incidentally, the type of nucleic acid probe to identify I contained can be suitably selected according to the kind of the target nucleic acid to be the subject, other types of RNA and the like nucleic acids may also be used. 当靶核酸为DNA时,识别探针I优选含有DNA ;当靶核酸为RNA时,识别探针I优选含有RNA。 When the target nucleic acid is a DNA, the DNA preferably contains a recognition probe I; when the target nucleic acid is RNA, preferably containing I recognition probe RNA. 当识别探针I含有RNA时,为了不使RNA被分解酶分解,也可使用被修饰的RNA例如寡核苷酸的2'位置取代为甲基的RNA。 When identifying RNA probe I contains, in order not to enzyme degradation is RNA, may be used, for example, modified RNA oligonucleotide 2 'position of the substituent is methyl RNA.

[0064] 关于DNAl〜DNA4的碱基长度,从合成及分析中操作方便的观点考虑,优选为10个碱基以上且1000个碱基以下,更优选为20个碱基以上且500个碱基以下。 [0064] About DNAl~DNA4 base length, to facilitate the analysis and synthesis from the operation viewpoint, preferably 10 bases or more and 1000 bases or less, more preferably 20 or more bases and 500 bases the following. 另外,在后述的核酸分析工序中为了防止DNAl〜DNA4对于作为靶的核酸的分析带来影响,并且能够充分地确保DNAl〜DNA4的定量性,在各容器2中所封入的DNAl〜DNA4的总分子数优选为100分子以上且100000分子以下。 Further, the later-described step of nucleic acid analysis in order to prevent DNAl~DNA4 impact analysis for a nucleic acid as a target, and sufficiently ensure the quantitative DNAl~DNA4 in each container 2 is sealed DNAl~DNA4 the total number of molecules is preferably 100 or more and 100,000 molecular molecule or less. 然而,毫无疑问根据核酸的分析方法的精度、灵敏度也可将识别探针I的总分子数设定在该范围以外。 However, there is no doubt that the nucleic acid analysis method according to the precision, and sensitivity can also be the total molecular recognition probe I is set outside this range.

[0065] 在对应表3中,作为各容器2内的识别探针I的组成即各识别探针I所含有的核酸的种类及其量,核酸的种类(DNA1〜DNA4)及其配混比与各容器2的容器号以I对I的方式进行对应。 [0065] 3 in the correspondence table, the type and amount of nucleic acid as a probe to identify the composition of I 2 in each of the containers to identify i.e. Probe I contained, the type of nucleic acid (DNA1~DNA4) and the mixing ratio each container 2 with the container in a manner I I of the correspondence.

[0066] 接着,对于使用以此方式构成的核酸分析试剂盒100的核酸分析方法,参照图2的流程图进行说明。 [0066] Next, using a nucleic acid configured in this manner nucleic acid analysis method of analyzing kit 100 will be described with reference to a flowchart of FIG.

[0067] 在使用本实施方式中的核酸分析试剂盒100对待检体中所含有的靶DNA进行分析时,首先将待检体投入至容器2中,在该容器2内使待检体与识别探针I充分地混合(步骤SI,探针添加工序)。 [0067] The target DNA nucleic acid analyte 100 to treat the specimen in the kit included in the present embodiment is used in the time of analysis, it is first to be specimen inserting into a container 2, in the inner 2 of the container to be subject to identification probe I thoroughly mixed (step SI, the probe addition step). 在此情况下,预先记录将哪个待检体投入到哪个容器号的容器2中(步骤S2) ο In this case, which will be recorded in advance into the specimen container which container No. 2 (step S2) ο

[0068] 其次,对各待检体实施用于核酸分析的前处理。 [0068] Next, a preprocessing for nucleic acid analysis for each specimen to be. 例如当待检体为生物组织的片段或细胞等时,进行从待检体提取DNA的处理。 For example, when the specimen is to be biological tissue fragments or cells, the process for extracting DNA from the specimen to be. 接着,通过使用定量的PCR等定量核酸扩增法对经前处理而由各待检体中得到的样品进行分析,从而对样品中所含有的靶DNA进行检测和定量(步骤S3,核酸分析工序)。 Subsequently, by using quantitative PCR and other nucleic acid quantitative analysis of nucleic acid amplification step of the method after preprocessing obtained by the sample in the specimen to be analyzed to target DNA contained in the sample is detected and quantified (step S3, the ).

[0069] 在此,被封入至容器2中的识别探针I也与源自待检体的靶DNA —起存在于各样品中。 [0069] Here, the probe is sealed to the container identification I 2 also be derived from a subject target DNA - from present in each sample. 对于构成识别探针I的4种DNAl〜DNA4而言,在步骤S3中也与靶DNA —起实施检测和定量。 DNAl~DNA4 configured for the 4 probe I is identified, in step S3, also the target DNA - detection and quantification of starting embodiment. 因此,4种DNAl〜DNA4具有通过靶DNA用的引物而扩增的碱基序列,或者在步骤S3中也添加DNAl〜DNA4用的引物。 Thus, four kinds DNAl~DNA4 having a base sequence of the amplified target DNA with the primers, or primers be added DNAl~DNA4 used in step S3. 由此,对于每个样品而言得到DNAl〜DNA4的配混比(步骤S4)。 Thus, for each sample, the mixing ratio DNAl~DNA4 obtained (Step S4).

[0070] 步骤S3的定量核酸扩增法中,优选以大致相同的扩增率对各样品中的DNAl〜DNA4进行扩增。 [0070] Step S3 quantitative nucleic acid amplification method, preferably in substantially the same expansion rate of each sample was amplified in DNAl~DNA4. 具体而言,DNAl〜DNA4的扩增率的差优选为1.9倍以下,更优选为1.1倍以下。 Specifically, the difference in amplification rates DNAl~DNA4 preferably 1.9 times or less, more preferably 1.1 times or less.

[0071 ] 接着,通过将在步骤S4得到的各样品的DNAl〜DNA4的配混比与对应表3相对照,从而能够特定各样品是源自于投入至哪个容器号的容器2中的待检体(步骤S5)。 [0071] Next, in step S4 by each sample obtained with a mixing ratio of 3 DNAl~DNA4 contrast to the correspondence table, it is possible to specify each input sample is derived from the number of containers into which container 2 to be tested body (step S5).

[0072] 如此,本实施方式通过对由各待检体所制备的样品中所含有的识别探针I进行分析,从而可鉴定该样品的来源。 [0072] Thus, the present embodiment by way of identification by the probe sample to be prepared in a specimen contained I analyzed, thereby identifying the source of the sample. 通常,在分子生物学实验中,从将待检体投入至容器2到靶DNA的分析结束为止的期间,从待检体或该待检体得到的样品被多次从一个容器转移至其他的容器。 Typically, the molecular biology experiments, the subject to be put to the container body 2 to the target DNA analysis period until the end, is to be repeatedly transferred from the specimen or sample to be obtained from one subject to another container container. 本实施方式具有如下优点:将样品进行转移时即便不记录将哪个样品移至哪个容器中,基于最终供于核酸分析的样品中所含有的识别探针I的配混比,也能够很准确地鉴定该样品的来源。 This embodiment has the following advantages: if not move to record which sample container which, based on the identification for the probe sample to a final nucleic acid analysis contained in the mixing ratio I when a sample is transferred, it is possible to very accurately identification of the source of the sample.

[0073] 进而还具有如下优点:通过使多种核酸的配混量不同,从而即使使用极少种类的核酸,也可以很容易地制造可相互识别的多种识别探针1,并能够鉴定多个样品来源。 [0073] Further also has the following advantages: more nucleic acids by blending different amounts, so that even if a few kinds of nucleic acids, can be manufactured more easily recognize the mutual identification probe 1, and capable of identifying multiple sample source.

[0074] 需要说明的是,在本实施方式中使用定量核酸扩增法进行核酸分析,但关于分析核酸的方法,只要可对样品中所含有的特定的核酸进行检测和定量即可,替代上述的定量核酸扩增法也可使用具有核酸定量功能的碱基序列读取装置(DNA测序)。 [0074] Incidentally, a nucleic acid analysis using quantitative nucleic acid amplification method in the present embodiment, but on the method for analyzing a nucleic acid, as long as a specific nucleic acid contained in the sample can be detected and quantified, instead of the quantitative nucleic acid amplification method can also read apparatus (DNA sequencing) using a nucleic acid having a nucleotide sequence of a quantitative function.

[0075] 即使以此方式进行,也可在对样品中所含有的靶DNA进行检测和定量的同时得到构成识别探针I的DNAl〜DNA4的配混比。 [0075] Even in this manner, it may be configured to obtain identification Probe I DNAl~DNA4 be compounded at the same time of the target DNA contained in the sample is detected and quantified.

[0076](第2实施方式) [0076] (Second Embodiment)

[0077] 接着,对于本发明的第2实施方式中的核酸分析试剂盒200及使用该试剂盒的核酸分析方法,参照图3至图5进行说明。 [0077] Next, a second embodiment of the present invention in nucleic acid analysis kit for nucleic acid analysis method and using the kit 200 of FIG. 3 to 5 will be described with reference to FIG.

[0078] 需要说明的是,在本实施方式中以与第I实施方式不同的点为主进行说明,对于与第I实施方式相同的构成以相同的符号标示并省略说明。 [0078] Incidentally, in the present embodiment and the first embodiment to embodiment I mainly different points will be described below, the same elements as in the first embodiment are labeled with the embodiment I the same reference numerals, and description thereof will be omitted.

[0079] 本实施方式中的核酸分析试剂盒200,如图3所示,如下点与第I实施方式不同,代替容器2而具备粘贴有多种识别探针I的基板4,在对应表3'中,各识别探针I的组成与其在基板4中的粘贴位置相对应。 [0079] The present embodiment is a nucleic acid assay kit 200, as shown in FIG. 3, the following points from the first embodiment different from embodiment I, instead of the container 2 and includes a substrate attached with a plurality of probes to identify the I 4, 3 in the correspondence table ', the composition of each probe I identified in its attaching position of the substrate 4, respectively.

[0080] 基板4是如盖玻片样的适用于粘贴从生物组织切出的切片的平板。 [0080] The substrate 4 is suitable for use in paste-like cover glass plate cut out sections from a biological tissue. 基板4设置有分割线4a,分割线4a将基板表面以棋盘的格状分割成多个区域。 The substrate 4 is provided with a dividing line 4a, 4a line dividing the surface of the substrate to form a checkerboard grid into a plurality of regions. 在通过该分割线4a分割的各区域中粘贴有I种识别探针1,识别探针I中所含有的DNAl〜DNA4的配混比在各区域中相互不同。 I-identification probes is attached in the respective regions divided by the dividing line 4a in 1, DNAl~DNA4 feature identification Probe I contained in the mixture ratio of the respective regions are different. 向基板4粘贴识别探针I时使用例如喷墨式打印机或用于DNA芯片制造的点样仪。 For example, ink jet printer or a DNA chip manufacturing spotter identifying probe I to 4 paste the substrate.

[0081] 在本实施方式中设想,与第I实施方式相同地,将4种DNA1、DNA2、DNA3和DNA4以4个阶段各自不同的配混量进行混合的情况。 [0081] contemplated in the present embodiment, in the first embodiment similarly to embodiment I, the four kinds of DNA1, DNA2, DNA3 and DNA4 to four phases respectively different blending amount of mixing conditions. 因此,通过分割线4a将基板4分割成256以下数目的区域即可。 Accordingly, the substrate 4a by dividing line 256 is divided into four regions can be the following number. 如图3所示的基板4被分割成10行X 10列并形成100个区域。 The substrate 34 shown in FIG. 10 is divided into a row and column X-10 are formed 100 regions.

[0082] 在对应表3'中,各区域的位置与在各区域所粘贴的识别探针I所具有的DNAl〜DNA4的配混比之间以I对I的方式进行对应。 [0082] In Table 3 the corresponding 'to be I I manner between a position corresponding to each region and in each region identified pasted probe I has a mixing ratio of DNAl~DNA4. 每个区域的位置使用行号a、b、c、…、j及列号A、B、…、J的方式进行特定。 The position of each region using the line number of a, b, c, ..., j and column number A, B, ..., J in a specific manner.

[0083] 接着,对于使用以此方式构成的核酸分析试剂盒200的核酸分析方法,参照图5的流程图进行说明。 [0083] Next, using a nucleic acid configured in this manner nucleic acid analysis Analysis of the kit 200, will be described with reference to the flowchart of FIG.

[0084] 在使用本实施方式中的核酸分析试剂盒200对生物组织的切片5中所含有的核酸进行分析时,如图4A所示,首先将从生物组织切出的切片5粘贴于基板4 (步骤S21,探针添加工序)。 [0084] In the present embodiment using a nucleic acid assay kit 5 200 pairs of sections of biological tissue for analysis contained in, as shown, from the biological tissue is first cut out sections attached to the substrate 4 4A 5 (step S21, the step of adding the probe). 接着,如图4B所示,沿着分割线4a切断基板4和切片5,从而得到在至少一部分的芯片上附着有待检体的切片5的片段的100个芯片(支撑体)4b (步骤S22,切断工序)。 Next, as shown in FIG. 4B, cutting the substrate 4 and 5 along the dividing line sections 4a, whereby a fragment of 100 chips 5 slices of at least a portion of the chip to be attached to the subject (a support member) 4B (step S22, the cutting step).

[0085] 对于切断基板4和切片5的方法没有特别限定,例如,通过将预先切断的芯片4b在可伸展的片上进行2维排列从而形成基板4,并将切片5粘贴于基板4后使片伸展并将芯片4b之间分开,从而能够沿着由芯片4b之间的界限所形成的分割线4a同时切断基板4和切片5。 [0085] For a method of cutting the substrate 4 and the slices 5 are not particularly limited, for example, by previously cutting the chip 4b two-dimensionally arrayed on the sheet can be stretched to form the substrate 4, and sections 5 attached to the substrate 4 after the sheet separation between the chip and stretch 4b, it is possible to simultaneously slicing and cutting the substrate 4 by the dividing line 5 along the boundary between the chip formed 4b 4a.

[0086] 得到的芯片4b分别例如投入至不同的微量离心管中,对于附着于芯片4b的切片5的片段进行了核酸提取等的前处理。 [0086] 4b are obtained, for example, into chips to a different microcentrifuge tube, for a slice segment 5 attached to the chip 4b were pretreated nucleic acid extraction and the like. 接着,将经前处理而从各待检体中得到的样品与第I实施方式的步骤S3相同地进行核酸分析,从而对样品中所含有的靶DNA进行检测和定量(步骤S23,核酸分析工序)。 Next, the pretreated obtained from each to be subject in the sample of step I of embodiment S3 in the same manner as nucleic acid analysis, such that the target DNA contained in the sample is detected and quantified (step S23, the nucleic acid analysis step ).

[0087] 在此,粘贴于基板4的I种识别探针I与源自片段的靶DNA—起存在于由切片5的各片段所制备的各样品中。 [0087] In this case, attached to the target species identification probes I I fragment derived from the substrate 4 from DNA- present in each sample was prepared from each segment of the 5 sections. 因此,以与上述步骤S3〜S5相同地方式,对于各样品,通过得到DNAl〜DNA4的配混比(步骤S24),且将得到的各样品的DNAl〜DNA4的配混比与对应表3'相对照,从而能够特定各样品是源自于哪个位置的片段(步骤S25)。 Thus, in the same manner as in the above-described step S3~S5, for each sample, obtained by the mixing ratio DNAl~DNA4 (step S24,), and the resulting DNAl~DNA4 feature mixing ratio of each sample with the corresponding table 3 ' in contrast, each sample can be derived from the particular segment in which position (step S25).

[0088] 本实施方式的效果与上述的第I实施方式相同,因此省略说明。 [0088] The effects of this embodiment I of the above-described embodiment, the description thereof will be omitted.

[0089] 需要说明的是,在本实施方式中,虽在基板4的各区域粘贴有识别探针1,但也可代替这种方式,在基板4上粘贴切片5后,使用上述的喷墨式打印机或点样仪等在切片5上粘贴识别探针I。 [0089] Incidentally, in the present embodiment, although the respective regions of the substrate 4 attached to the probe 1 with an identification, but also replace this manner, after pasting slices 5 on the substrate 4, the above-described ink jet printer or the like attached spotter I. probes identified in the slice 5 在此情况下,以通过分割线4a所分割的各区域各含有一种识别探针I的方式也能决定识别探针I的粘贴位置。 In this case, each of the divided region division line 4a of each probe I contains a recognition decision manner the attaching position can be identified by the I probe.

[0090]即使这样做也能基于各样品中所含有的识别探针I的组成来准确地特定供于核酸分析的各样品分别是源自于切片5的哪个位置的片段。 [0090] even if it can be identified based on the composition of each sample Probe I contained accurately specific to each sample for analysis of nucleic acid fragments which are derived from the 5 position of the slice.

[0091](第3实施方式) [0091] (Third Embodiment)

[0092] 接着,对于本发明的第3实施方式中的核酸分析试剂盒及使用该核酸分析试剂盒的核酸分析方法,参照图6进行说明。 [0092] Next, a third embodiment of the present invention in a nucleic acid assay kit and nucleic acid analysis using the nucleic acid analysis method of the kit, will be described with reference to FIG. 本实施方式涉及使用第2实施方式中的核酸分析试剂盒200的另一个核酸分析方法。 The present embodiment relates to a further embodiment nucleic acid analysis method using the second embodiment, a nucleic acid assay kit 200. 因此,对于与第2实施方式不同的方面主要进行说明,对于与第2实施方式相同的构成用相同的符号标示并省略说明。 Thus, those of the second embodiment different aspects will be mainly described, those of the second embodiment configured in the same manner, and designated by the same reference numerals will be omitted.

[0093] 根据本实施方式中的核酸分析方法,如图6所示,步骤S21中将切片5粘贴于基板4上后,通过原位杂交在切片5上使检测用的DNA探针(核酸探针。以下称为检测用探针。)与作为靶的RNA缔合(步骤S31,杂交工序)。 [0093] The nucleic acid analysis method according to the present embodiment, as shown in FIG. 6, after the step S21 will be attached to sections 5 on the substrate 4, by in situ hybridization detection of the DNA probe (nucleic acid probe in the slice 5 needle hereinafter referred to as a detection probe.) RNA associated with a target (step S31, the hybridization step). 使检测用探针缔合后,与第2实施方式相同地分割并提取切片5 (步骤S22),并进行核酸分析(步骤S23),取得识别探针I的配混比(步骤S24),基于对应表3'来鉴定待检体(步骤S25)。 After the detection probe associated, similarly to the second embodiment is divided and extracted sections 5 (step S22), and nucleic acid analysis (step S23), identifying the probe I to obtain the mixing ratio (step S24,), based on 3 'of the subject to be identified (step S25) correspondence table. 然而,在本实施方式中,在步骤S23中对检测用探针也进行分析,在步骤S24中也能取得各待检体中所含有的检测用探针的种类和量。 However, in the present embodiment, in Step S23 the detection probe analysis also can be obtained when the kind and amount of each of the detection probe to be contained in the subject in step S24.

[0094] 如果不使用预先粘贴有识别探针I的基板4,而在基板4上粘贴切片5后使用喂■墨式打印机等粘贴识别探针I的情况下,在步骤S31的杂交之后将识别探针I粘贴于切片5。 A case where the [0094] If you do not use pre-pasted I recognition probe substrate 4, and 5 sections were attached on the substrate 4 fed ■ paste like ink printer identification I of probe, the hybridization step S31 after the identification a probe attached to the slice I 5.

[0095] 根据本实施方式中的核酸分析方法,除了第2实施方式的效果以外,在用定量核酸扩增法对靶核酸进行解析的情况下,通过使用已知的检测用探针可使扩增效率大致相同,而且可提高检测的精度。 [0095] The nucleic acid analysis method according to the present embodiment, in addition to the effects of the second embodiment, in the case of analyzing a target nucleic acid amplification method of quantitative nucleic acid, by using a known detection probe can expand by substantially the same efficiency, but also improve the accuracy of detection. 另外,特别是靶核酸为RNA时,在进行核酸分析时能够不将RNA转换为互补的DNA而通过定量核酸扩增法和DNA测序来进行解析。 Further, especially when the target nucleic acid RNA, the RNA can not be converted to the complementary DNA analyzing method and DNA sequencing amplified nucleic acid during nucleic acid by quantitative analysis. 而且,通过将易分解的RNA置换为DNA来进行检测,可以提高检测的精度和灵敏度。 Further, to detect the RNA by replacement of the easily decomposed be DNA, can improve the accuracy and sensitivity of detection.

[0096] 需要说明的是,在上述的第I〜第3的实施方式中,对于含有4种核酸的识别探针I进行了说明,但识别探针I中所含有的核酸的种类的数目不限于这些,可根据实际需要选择合适的数目。 [0096] Incidentally, in the above-described first embodiment I~ third embodiment, a probe for identifying a nucleic acid containing four kinds of I has been described, but the number identifying the type of probe I is not contained in a nucleic acid limited thereto, according to actual needs to select the appropriate number. 例如,在第2实施方式中,优选为适用于喷墨式打印机或点样仪的数目。 For example, in the second embodiment, it is preferred to apply to the number of ink jet type printer or a dot-like instrument. 具体地,识别探针I所含有的核酸的种类的数目,在使用4色用喷墨式打印机时,优选核酸种类的数目为4以下,使用点样仪时,优选为点样仪所配备的针的数目以下。 Specifically, the identification number of the kinds of probe nucleic acids contained in I, when used with a four-color inkjet printer, the number of nucleic acid species is preferably 4 or less, when using a spotter, and preferably is equipped with a spotter the number of needles or less.

[0097] 另外,在上述的第I〜第3的实施方式中,为了通过核酸分析而准确地得到DNA1、DNA2、DNA3和DNA4的配混比,对于DNA1、DNA2、DNA3和DNA4的各自的配混量而言,当最小的配混量作为I时,以2倍、3倍、4倍不同的方式进行设定。 [0097] Further, in the above-described first embodiment I~ third embodiment, in order to accurately by nucleic acid analysis to give DNA1, DNA2, DNA3, and mixing ratio with DNA4 for DNA1, DNA2, DNA3, and with each of DNA4 the compounding amount, when the minimum compounding amount as I, 2-fold, 3-fold, 4-fold is set in different ways. 然而,能够以高精度检测样品中所含有的DNAl、DNA2、DNA3和DNA4的量时,也可以使DNAl、DNA2、DNA3和DNA4各自的配混量以更小的比率进行变化。 However, the sample can be detected with high precision DNAl contained, DNA2, DNA3 and the amount DNA4 also possible to DNAl, DNA2, DNA3 and DNA4 compounding amount of each of a smaller ratio changes.

[0098] 另外,在上述的第I〜第3的实施方式中,识别探针I虽含有多种核酸,但根据分析对象的待检体的数目或核酸分析的定量精度等条件,识别探针I也可仅含有I种核酸。 [0098] Further, in the above-described first embodiment I~ 3, identifying a nucleic acid probe containing a variety of I though, but quantitative analysis accuracy depending on the number or nucleic acid sample to be analyzed target conditions to identify probes I also may contain only I nucleic acids.

[0099] S卩,在以第I实施方式为例进行列举时,核酸分析试剂盒100在具备η个(η为自然数。)容器2时,各容器2中封入仅含有I种核酸的识别探针1,并使该核酸的量在每个容器2中η级不同。 [0099] S Jie, when in the first I embodiment example include, nucleic acid analysis kit 100 includes a [eta] a ([eta] is a natural number.) When the container 2, each container is filled only contains identification Probe I nucleic acid in 2 pin 1, and the amount of the nucleic acid in each container 2 in a different stage η.

[0100] 即使这样做,通过对供于核酸分析的样品中的识别探针I进行分析,从而可以鉴定样品的来源。 [0100] Even if doing so, by analyzing the samples for the analysis of nucleic acid probes to identify the I, thereby identifying the source of the sample.

[0101] 附图标记说曰月 [0101] Reference numeral said said month

[0102] 100,200核酸分析试剂盒 [0102] The nucleic acid analysis kit 100,200

[0103] I识别探针 [0103] I recognition probe

[0104] 2容器(支撑体) [0104] 2 container (support)

[0105] 3,3' 对应表 [0105] 3,3 'correspondence table

[0106] 4 基板 [0106] 4 substrate

[0107] 4a分割线 [0107] 4a parting line

[0108] 4b芯片(支撑体) [0108] 4b chip (support)

[0109] 5 切片 [0109] 5 slices

[0110] S1,S21探针添加工序 [0110] S1, S21 step of adding the probe

[0111] S3,S23核酸分析工序 [0111] S3, S23 nucleic acid analysis step

[0112] S22切断工序 [0112] S22 cutting step

[0113] S31杂交工序 [0113] S31 hybridization step

Claims (18)

1.一种核酸分析试剂盒,其具备多个支撑体,所述多个支撑体保持含有核酸的识别探针且支撑待检体, 所述识别探针包含具有已知的碱基序列的至少I种核酸,且在每个所述支撑体中的所述核酸的种类和量中的至少一者不同并以能够混合于所述待检体的方式保持在所述支撑体中。 A nucleic acid analysis kit comprising a plurality of supports, the plurality of support holder comprising a nucleic acid probe to identify and support member to be detected, the probe comprises at least identification of the base sequence with known I nucleic acid, and at least one of a different kind and amount of the nucleic acid in each of the support body in a manner and can be mixed with the specimen to be held in the support body.
2.根据权利要求1所述的核酸分析试剂盒,其具备对应表,所述对应表将所述多个支撑体与分别被该多个支撑体保持的所述识别探针的所述核酸的种类和量的组合相对应。 The nucleic acid assay kit according to claim 1, which includes a correspondence table, said correspondence table of said plurality of said support and said probe are identified holding the plurality of supporting nucleic acid the kind and amount of the combination, respectively.
3.根据权利要求1或权利要求2所述的核酸分析试剂盒,其中, 所述支撑体为收容所述待检体的容器, 所述识别探针被封入所述容器内。 3. The nucleic acid according to claim 2 or assay kit as claimed in claim 1, wherein the support is a container accommodating the specimen to be, the identification probe is enclosed within said container.
4.根据权利要求1或权利要求2所述的核酸分析试剂盒,其具备基板, 所述基板能够沿着规定的分割线分割成多个芯片且粘贴有作为所述待检体的生物组织的切片, 所述识别探针分别被所述多个芯片保持。 According to claim 1 or a nucleic acid assay kit according to claim 2, comprising a substrate along a predetermined dividing line can be divided into a plurality of chips and is attached to be used as a biological tissue specimen slice, the identification of the plurality of probe chips retaining respectively.
5.根据权利要求1〜权利要求4中任一项所述的核酸分析试剂盒,其中,所述核酸为DNA0 According to any of claim 1 ~ 4, wherein a nucleic acid assay kit of any one of claims, wherein said nucleic acid is DNA0
6.根据权利要求5所述的核酸分析试剂盒,其中,所述DNA的碱基长度为20个碱基以上且500个碱基以下。 The nucleic acid of claim 6. The assay kit of claim 5, wherein said DNA base length of 20 bases or more and 500 or less bases.
7.根据权利要求5或权利要求6所述的核酸分析试剂盒,其中,保持在各所述支撑体的所述DNA的分子数为100分子以上且100000分子以下。 According to claim 5 or claim 6, wherein said nucleic acid assay kit, which is maintained at the number of molecules of the DNA of each of the support is 100 or more and 100,000 molecular molecule or less.
8.—种核酸分析方法,其中包括以下工序: 探针添加工序:分别向多个待检体中添加包含具有已知的碱基序列的至少I种核酸的识别探针,及核酸分析工序:对各所述待检体中所含有的核酸进行分析, 所述探针添加工序中,将所述核酸的种类和量中的至少一者相互不同的所述识别探针向所述多个待检体中添加, 所述核酸分析工序中,对于向各所述待检体中添加的所述识别探针中所含有的所述核酸也进行解析。 8.- nucleic acid analysis methods, including the following steps: a step of adding a probe: a plurality respectively to be added to the specimen containing at least I-identification probe nucleic acid, and nucleic acid analysis process having a known base sequence: of each of the nucleic acid analysis to be contained in the specimen, the probe is added in step, the kind and amount of the nucleic acid probe to identify at least one of each of said plurality to be different from adding the sample, the nucleic acid analysis step for identifying the nucleic acid probe in each of the specimen to be added is also contained in the parsed.
9.根据权利要求8所述的核酸分析方法,其中,所述探针添加工序中,将所述待检体投入至收容所述识别探针的容器内。 9. The nucleic acid analysis method according to claim 8, wherein the step of adding the probe, the specimen to be put into the container housing the probe identification.
10.根据权利要求8所述的核酸分析方法,其中, 所述探针添加工序中,在散在并粘贴有所述识别探针的基板上粘贴生物组织的切片, 进而包括切断工序:将粘贴有所述切片的基板与所述切片一起切断成含有一个识别探针的多个芯片, 所述核酸分析工序中,对于附着于在所述切断工序中得到的所述芯片上的所述切片的一部分进行分析。 10. The nucleic acid analysis method according to claim 8, wherein the step of adding the probe, and is attached in the bulk of the biological tissue identification sections attached on the substrate of the probe, further comprising a cutting step of: pasted the substrate and the slice with the slice cut into a plurality of chips comprising a probe to identify the nucleic acid analysis step, the upper is attached to the chip to obtain the cutting step in part of the slice for analysis.
11.根据权利要求8所述的核酸分析方法,其中, 所述探针添加工序中,在生物组织的切片上散在并粘贴所述识别探针, 进而包括切断工序:将粘贴有所述识别探针的所述切片切断成含有一个识别探针的多个片段, 所述核酸分析工序中,对于在所述切断工序中切断的所述切片的片段进行分析。 11. The nucleic acid analysis method according to claim 8, wherein the step of adding the probe, the biological tissue in the slice and paste dispersed in the identification probe, further comprising a cutting step of: identifying said probe is attached the slice is cut into a plurality of needle fragment containing a probe to identify the nucleic acid analysis step, for the segment of the slice is cut in the cutting step for analysis.
12.根据权利要求10或权利要求11所述的核酸分析方法,其还包括杂交工序:在所述切断工序前,使用具有与靶核酸互补的已知的碱基序列的核酸探针对于所述切片进行原位杂交。 12. A nucleic acid analysis method according to claim 11 or claim 10, further comprising a hybridization step of: prior to said cutting step, known nucleic acid probe having a base sequence complementary to the target nucleic acid to the slice situ hybridization.
13.根据权利要求8〜权利要求12中任一项所述的核酸分析方法,其中,所述核酸分析工序中,使用定量核酸扩增法或能够进行核酸定量的碱基序列读取装置对所述待检体及所述识别探针中所含有的核酸进行分析。 Claim according to claim 8~ nucleic acid analysis method according to any of claims 12, wherein the step of nucleic acid analysis, or quantitative nucleic acid amplification method capable of quantitative nucleic acid base sequence of the reading device said body to be examined to identify the probe and nucleic acid contained in the analysis.
14.根据权利要求8〜权利要求13中任一项所述的核酸分析方法,其中,所述识别探针中所含有的所述核酸为DNA。 14. The nucleic acid analysis method according to any one of claims 13, wherein said identifying the nucleic acid probe is DNA contained in claim 8~ claimed.
15.根据权利要求14所述的核酸分析方法,其中,所述DNA的碱基长度为20个碱基以上且500个碱基以下。 15. The nucleic acid analysis method according to claim 14, wherein said DNA base length of 20 bases or more and 500 or less bases.
16.根据权利要求14或权利要求15所述的核酸分析方法,其中,各所述识别探针中所含有的所述DNA的分子数为100分子以上且100000分子以下。 16. A nucleic acid analysis method according to claim 15 or claim 14, wherein the number of molecules of each of the DNA probes contained in the identification of molecules of 100 or more and 100,000 or less molecule.
17.根据权利要求14〜权利要求16中任一项所述的核酸分析方法,其中, 所述核酸分析工序中,使用定量核酸扩增法对添加于各所述待检体中的所述DNA进行分析, 所述定量核酸扩增法中的所述DNA的扩增率大致相同。 17. The claim as claimed in claim 14~ nucleic acid analysis method according to any one of 16, wherein said nucleic acid analysis step, a quantitative nucleic acid amplification method to be added to each of the specimen in the DNA analysis, the quantitative amplification of the DNA in the nucleic acid amplification method is substantially the same.
18.根据权利要求17所述的核酸分析方法,其中, 所述核酸分析工序中,使用PCR法对添加于各所述待检体中的所述DNA进行分析, 所述PCR法中的各所述DNA的扩增率的差为1.9倍以下。 18. The nucleic acid analysis method according to claim 17, wherein the step of nucleic acid analysis, using the PCR method to be added to each of the specimen in the analysis of DNA, as in each of the PCR method said difference amplified DNA ratio of 1.9 times or less.
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